Importance of Rare DPYD Genetic Polymorphisms for 5-Fluorouracil Therapy in the Japanese Population

Abstract

Dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is the rate-limiting enzyme in 5-fluorouracil (5-FU) degradation. In Caucasians, four DPYD risk variants are recognized to be responsible for interindividual variations in the development of 5-FU toxicity. However, these risk variants have not been identified in Asian populations. Recently, 41 DPYD allelic variants, including 15 novel single nucleotide variants, were identified in 3,554 Japanese individuals by analyzing their whole-genome sequences; however, the effects of these variants on DPD enzymatic activity remain unknown. In the present study, an in vitro analysis was performed on 41 DPD allelic variants and three DPD risk variants to elucidate the changes in enzymatic activity. Wild-type and 44 DPD-variant proteins were heterologously expressed in 293FT cells. DPD expression levels and dimerization of DPD were determined by immunoblotting after SDS-PAGE and blue native PAGE, respectively. The enzymatic activity of DPD was evaluated by quantification of dihydro-5-FU, a metabolite of 5-FU, using high-performance liquid chromatography-tandem mass spectrometry. Moreover, we used 3D simulation modeling to analyze the effect of amino acid substitutions on the conformation of DPD. Among the 41 DPD variants, seven exhibited drastically decreased intrinsic clearance (CL _int ) compared to the wild-type protein. Moreover, R353C and G926V exhibited no enzymatic activity, and the band patterns observed in the immunoblots after blue native PAGE indicated that DPD dimerization is required for its enzymatic activity. Our data suggest that these variants may contribute to the significant inter-individual variability observed in the pharmacokinetics and pharmacodynamics of 5-FU. In our study, nine DPD variants exhibited drastically decreased or no enzymatic activity due to dimerization inhibition or conformational changes in each domain. Especially, the rare DPYD variants, although at very low frequencies, may serve as important pharmacogenomic markers associated with the severe 5-FU toxicity in Japanese population.

Keywords: 5-fluorouracil; DPYD; dihydropyrimidine dehydrogenase (DPD); genetic polymorphism; pharmacogenomics.

FIGURE 1

Metabolic pathway of 5-fluorouracil and uracil. 5-Fluorouracil and uracil are catabolized by dihydropyrimidine dehydrogenase, dihydropyrimidinase, and β-ureidopropionase.

FIGURE 2

Expression levels of wild-type and variant DPD proteins. (A) DPD protein levels were determined by immunoblotting analysis following SDS-PAGE. (B) DPD units of proteins expressed in 293FT cells. Each bar represents the mean ± standard deviation of three independent assays.

FIGURE 3

Michaelis-Menten curves of DPD variants. The kinetic parameters K _m , V _max , and CL _int of 5-FU reduction were determined. The velocities for the reduction of 5-FU and 5-FU concentration are plotted on the horizontal and vertical axes, respectively.

FIGURE 4

Immunoblotting analysis after blue native PAGE showing immunoreactive dihydropyrimidine dehydrogenase (DPD) variant proteins. Blue native PAGE was performed using Tris-glycine buffer and 5%–20% polyacrylamide gels; 9 μg of the soluble fraction of DPD variant proteins were loaded into each lane in triplicate. DPD variants were detected using polyclonal antibodies against human DPD.

FIGURE 5

Dihydropyrimidine dehydrogenase (DPD) structural analysis. (A) Diagram showing overall structure of human DPD. (B) Diagram showing each domain of human DPD.

FIGURE 6

DPD structural analysis. (A) Diagram of a fragment of the crystal structures of the H25R variant. The R25 residues are shown in yellow. (B) Diagram of a fragment of the crystal structures of the H304Y variant. The Y304 residues are shown in yellow. (C) Diagram of a fragment of the crystal structures of the F438V variant. The F438 residues are shown in yellow. (D) Diagrams of a fragment of the crystal structures of wild-type DPD (left panel) and R353C (right panel). The R353 and C353 residues are shown in yellow. Yellow and blue lines indicate hydrogen bonds and hydrophobic interactions, respectively.